William J. Rutter

An ultrastructural analysis of the developing embryonic pancreas

This ultrastructural analysis of pancreas development under in vivo and in vitro conditions complements earlier biochemical studies. The endocrine and exocrine tissue develops similarly in vitro. The early pancreatic epithelial cells, like those of the gut, form a single layer of cells facing the lumen, and are linked together by junctional complexes. This cellular polarization implies a constraint for the axis of division of exocrine cells, and demands that the endocrine cells loose their connection with the exocrine cells in order to form islets. The presence of mitotic figures in well differentiated acinar cells suggests that cell multiplication is not incompatible with cell differentiation. The first zymogen granules that can be observed look similar to the granules present later in development. This observation suggests that the “prozymogen granules” which were reported in earlier papers were probably the result of fixation artifacts, and did not represent a stage in granule formation between the condensing vacuoles in the Golgi and the definitive zymogen granules. The morphological observations of this study are consistent with the previously proposed biphasic model of differentiation for the exocrine and endocrine B cells. The development of specific organelles correlates with the pattern of accumulation of the specific exocrine proteins and insulin. On the other hand, differentiated A cells are present when the pancreatic bud first forms. This early appearance of differentiated A cells suggests a regulatory role for this hormone in early development.

Heterogeneity of Presumably Homogeneous Protein Preparations

Some highly purified glycolytic enzymes have been subjected to isoelectric focusing and found to contain a number of enzymatically active species. Crystalline aldolase A and glyceraldehyde-3-phosphate dehydrogenase from rabbit muscle were resolved into five components, crystalline aldolase from yeast was resolved into three components, pyruvate kinase from rabbit muscle yielded four components, and yeast enolase was resolved into two components. Rabbit muscle lactate dehydrogenase (M4) gave one major peak of protein and enzymatic activity. The profiles of aldolase, glyceraldehyde-3-phosphate dehydrogenase, and yeast aldolases suggest random combinations of two closely related subunits into tetramers and dimers, respectively. The molecular heterogeneity of the other enzymes is not so easily related to subunit structure.

Effects of a partially purified factor from chick embryos on macromolecular synthesis of embryonic pancreatic epithelia.

Abstract Essentially normal development of early embryonic pancreatic epithelium occurs only in the presence of mesenchymal tissues ( Golosow and Grobstein, 1962 ), or a particulate fraction (MF) obtained from extracts of chicken embryos (Rutter et al. , 1964). We have shown that this fraction also stimulates the incorporation of thymidine- 3 H into DNA. This stimulatory activity was detected in particulate fractions from homogenates of several mesodermal tissues from rat and chick embryos, as well as in fibroblasts cultured from these tissues, but not in embryonic epithelial tissues. This activity may thus be related to the mesodermal tissue requirement for pancreatic development. MF was solubilized and partially purified from homogenates of chick embryos. It is stable to collagenase, hyaluronidase, and neuraminidase. Activity is lost by heating and by treatment with trypsin. It is presumed, therefore, that the factor is associated with a protein that is not collagen. The effects of the MF upon macromolecular synthesis were tested in pancreatic tissues from 12-day rat embryos. When isolated epithelia were cultured in the absence of mesoderm or MF, the rate of thymidine- 3 H incorporation into DNA decreased to low levels. The specific activities of DNA polymerase and deoxycytidylate deaminase in epithelial extracts also declined. In contrast, the rate of thymidine- 3 H incorporation into DNA increased 5- to 8-fold over the initial rates in epithelia cultured with MF. Concurrently DNA polymerase activity in tissue extracts increased by 2- to 3-fold; deoxycytidylate deaminase activity declined slightly. MF also affected RNA and protein synthesis. The rate of leucine- 3 H incorporation into protein and uridine- 14 C incorporation into RNA in isolated pancreatic epithelia was comparable to that of intact rudiments. Cultures in the presence of MF increased these rates severalfold after 20 hr. These results suggest that MF, and by implication, mesoderm, may supply a growth factor for epithelial tissue and thus serves a permissive rather than a determining role in the differentiation process in pancreatic development.

Glyceraldehyde-3-Phosphate Dehydrogenase Variants in Phyletically Diverse Organisms

Electrophoretically distinct forms of glyceraldehyde-3-phosphate dehydrogenase (TDH) have been detected in turtle, perch, trout, spinach, and yeast. Multiple forms were not detected in rat, rabbit, chicken, frog, honey bee, Euglena, or Escherichia coli. The combination of two different subunits into tetramers is a probable explanation for the five-membered sets usually detected in extracts exhibiting TDH multiplicity.

Synthesis and accumulation of insulin in the fetal rat pancreas.

A micromodification of the double-antibody immunoassay for insulin is presented which can readily detect 106 molecules of standard insulin. The assay also measures proinsulin and related molecules. Insulin and immunologically related molecules are present at the time of the initial appearance of the pancreatic primoridum (11 days). Insulin-specific immunoreactivity in the pancreas rises from an undetectable level (<1 molecule/cell) at 10.5 days (15–17 somites) to a low but significant level at 12 days (35–40 somites) which remains constant until day 14. Specific immunoreactivity then rises 200-fold to the level characteristic of the postnatal pancreas. Normal cytodifferentiation of explanted 12-day pancreatic rudiments was obtained in organ culture, and the biphasic pattern of insulin accumulation seen in vivo was reproduced in vitro. Synthesis of insulin and related molecules was demonstrated during the period between 12 and 14 days.

Induction of tyrosine aminotransferase by triamcinolone during rat liver development in vitro

Abstract Embryonic rat liver 12–21 days old exhibits low but significant tyrosine aminotransferase (TAT) activity. In organ culture for 24 hr in a nutrient medium, there is an increase in TAT levels. Addition of glucocorticoids increases TAT levels at all embryonic ages. The magnitude of the produced TAT level increases with developmental age. Glucagon also increases embryonic liver TAT, but insulin and growth hormone (somatotropin) had little effect.